Cryopreservation of Hematopoietic Stem Cells: Emerging Assays, Cryoprotectant Agents, and Technology to Improve Outcomes

Abstract

Hematopoietic stem cell (HSC) therapy is widely used to treat a growing number of hematological and non-hematological diseases. Cryopreservation of HSCs allows for cells to be transported from the site of processing to the site of clinical use, creates a larger window of time in which cells can be administered to patients, and allows sufficient time for quality control and regulatory testing. Currently, HSCs and other cell therapies conform to the same cryopreservation techniques as cells used for research purposes: cells are cryopreserved in dimethyl sulfoxide (DMSO) at a slow cooling rate. As a result, HSC therapy can result in numerous adverse symptoms in patients due to the infusion of DMSO. Efforts are being made to improve the cryopreservation of HSCs for clinical use. This review discusses advances in the cryopreservation of HSCs from 2007 to the present. The preclinical development of new cryoprotectants and new technology to eliminate cryoprotectants after thawing are discussed in detail. Additional cryopreservation considerations are included, such as cooling rate, storage temperature, and cell concentration. Preclinical cell assessment and quality control are discussed, as well as clinical studies from the past decade that focus on new cryopreservation protocols to improve patient outcomes.

Cryopreservation as a Key Element in the Successful Delivery of Cell-Based Therapies—A Review

“…Any potentially cytotoxic effects of cryoprotectants, especially DMSO, will be reduced by lowered temperature and the avoidance of abrupt temperature change. Consequently, working temperatures in the range 0–4°C are beneficial ( 15 , 73 , 75 ) and practices such as adding cooled protectant to precooled cells or warmer protectant to warmer cells with immediate subsequent cooling are to be recommended ( 114 – 116 ).…”

“…Sample volume reduction, achieved by increasing cell concentration, can be attractive as it will limit materials and reagents used, processing time and cryostorage space as well as reducing the quantity of DMSO infused to the patient ( 11 , 117 – 119 ). However, cell concentrations higher than ~200 × 10 6 cells/mL appear detrimental on engraftment prediction (through the CFU-GM assay) or engraftment yield post-thaw ( 88 , 114 , 117 , 118 , 120 , 121 ). As the ice fraction within a cooling sample grows and the unfrozen channels, where cells are confined, reduce in size there will be an increase in potentially injurious compression forces and direct cell to cell contact ( 11 ).…”

De-risking the final formulation, fill and finish step in cell therapy manufacturing: considerations for an automated solution

“…In addition, the automated system should allow process controls for temperature and the rate of cryoprotectant addition. Reiterating the point, the addition of cryoprotectant such as DMSO, to a cell suspension is an exothermic reaction and the release of energy could damage the cells [6,12]. Considering this significant need for process controls and slow addition of pre-cooled cryoprotectant, an automated system must provide controlled-rate addition of cryoprotectant and should monitor the temperature throughout the formulation and fill process.…”

Evaluation of the effectiveness of a new cryopreservation system based on a two-compartment vial for the cryopreservation of cell therapy products

“…Dimethyl sulfoxide (Me 2 SO) is the most widely used cryoprotectant for cell therapy [12]; however, it is known to induce toxicity and epigenetic changes in cells [13,14]. Also, numerous studies have reported Me 2 SO-related adverse reactions following infusion of Me 2 SO-preserved cell products, ranging from mild events such as nausea, vomiting, headache, hypotension, hypertension and diarrhea to severe reactions such as cardiac arrhythmias, cardiac arrest, respiratory stress and epileptic seizures [5,11,13,15,16]. Thus, it would be desirable to reduce the levels of Me 2 SO (e.g., from 10% to 5% [17,18]) in cellular products for clinical use both in final and intermediate products.…”

“…Developing new optimized protocols, vials and devices that improve cell cryopreservation/thawing and allow standardization could increase the cellular recovery and functionality of cryopreserved cells, ensuring their effective clinical application, ultimately benefiting the patient [29]. Although considerable progress has been made in optimizing freezing protocols, freezing media composition, cooling devices and cryovials to ensure that cell products are safe and retain their therapeutic characteristics following cryopreservation, more research focused on improving cell recovery and Me 2 SO-associated injury is still needed [5,11].…”